N,N&#39;-divinylalkylurea crosslinked polymers, a process for their preparation, and their use

ABSTRACT

The invention relates to crosslinked polymers based on polyvinyl acylate or polyvinyl alcohol and having crosslinking agent units which are derived from crosslinking agents of the general formulae ##STR1## and/or ##STR2## These products, inter alia, are particularly hydrolysis-resistant and can have high crosslinking densities. 
     The invention also relates to a process for preparing these polymers and to their use in chromatography and as carriers for biologically active substances.

This application is a continuation of application Ser. No. 898,749,filed Aug. 18, 1986 which was, in turn, a continuation of applicationSer. No. 680,461 filed Dec. 11, 1984, both now abandoned.

The use of polymer gels for gel permeation chromatography of polymersolutions for the purpose of separating or purifying substances or fordetermining the molecular weight distribution has been known quite along time. Polymer gels which are suitable for aqueous systems arereferred to as hydrophilic, whereas those which can only be used innon-aqueous systems (organic solvents) are referred to as hydrophobic.Examples of hydrophobic gels are crosslinked polystyrenes, whereashydrophilic gels are based on crosslinked dextrans,polyvinylpyrrolidone, polyacrylamide or polyvinyl alcohol. Any excessivetendency of these gels to swell and hence, for example, prevent highflow rates in gel permeation chromatography can be counteracted byincreasing the degree of crosslinking.

There are already existing hydrophobic gels based on crosslinkedpolyvinyl acetate from which, by hydrolyzing the acetate groups, ahydrophilic gel based on crosslinked polyvinyl alcohol can be prepared.Here the important point, above all, is that the crosslinking must behighly resistant to hydrolysis.

A number of compounds have already been described as crosslinking agentsfor this purpose in the prior art. For instance, German Pat. No.1,517,935 discloses for this purpose not only divinylalkylenes, divinyland dialkyl esters of dicarboxylic acids and others but also divinyl ordiallyl ethers of polyhydric alcohols, preference being given tobutanediol divinyl ether (cf. in this context also Makromol. Chemie 176,pages 657 et seq. (1975)). The crosslinked polyvinyl acetates andpolyvinyl alcohols which can be obtained according to said patent canalso be in the form of macroporous beads. It is true that thecrosslinking with butanediol divinyl ether is stable to hydrolysis, butthis ether copolymerizes with vinyl acetate only relatively reluctantly,so that only relatively low crosslinking densities can be obtained withthis crosslinking agent.

Other existing crosslinking agents such as ethylene glycol dimethylacrylate or glycidyl methacrylate (U.S. Pat. No. 4,104,208) do notproduce hydrolysis-resistant crosslinking and are not incorporateduniformly. Thus, the copolymerization parameters of vinyl acetate (M₁)and methyl methacrylate (M₂) are r₁ =0.01 r₂ =20. Similarcopolymerization parameters are likely for the methacrylates mentionedhere. So the two crosslinking agents mentioned are consumed at the startof the reaction; it is therefore impossible to obtain uniformincorporation.

It is also already part of the state of the art to use hydrophilicpolymer gels for affinity chromatography for separating biologicallyactive substances, i.e. for immobilizing such substances, and beforethis use to react the reactive groups of the polymer gel, which areusually OH groups, with so-called spacers. Spacers in this sense includeinter alia epichlorohydrin (cf. German Offenlegungsschrift 2,102,514 andGerman Pat. No. 2,421,789).

It is then the object of the present invention to provide a crosslinkedpolymer which is based on polyvinyl ester and in particular polyvinylalcohol, which does not have the state of the art disadvantages, which,in particular is hydrolysis-resistant and is particularly suitable foruse as an adsorbent in gel chromatography or as a carrier material forchemically covalently bonded biologically active substances, and whichhardly impairs the activity of the chemically covalently bonded,biologically active substances and guarantees unimpeded flow of thesubstrates to be treated.

The invention accordingly relates to a crosslinked polymer whichessentially comprises vinyl acylate units and units of a crosslinkingagent which has the general formulae ##STR3## and/or ##STR4## where R₁and R₂ in the formula (I) can be identical or different and each denotesvinyl, 1-acyloxyvinyl, allyl or 2-acyloxyallyl, A represents a divalenthydrocarbon radical of 2 to 8 carbon atoms, B in the formula (II) standsfor a divalent, trivalent or tetravalent hydrocarbon radical of 1 to 8carbon atoms, m corresponds to the valency of this radical and X denotesacyloxy, the amount of these crosslinking agent units accounting for 0.1to 60% by weight of the polymer.

Preferably, the acylate groups of the vinyl acylate units are partiallyor completely replaced by OH groups.

The invention also provides a process for preparing this crosslinkedpolymer by copolymerizing vinyl acylate with a crosslinking agent in thepresence of a dispersion medium wherein the crosslinking agent has theabove formulae (I) and/or (II). Preferably, the polymer thus obtained issubsequently partially or completely hydrolyzed.

Finally, the invention also concerns the use of the polymers accordingto the invention as adsorbents in chromatography or as carrier materialsfor biologically active substances.

The vinyl acylate units of the polymer according to the inventionpreferably contain 2 to 18 carbon atoms, in particular 2 to 6 carbonatoms, in the acylate radical. The latter is preferably an acetate orpropionate radical. It is also possible for various acylate radicals tobe present in the polymer, which is to say that the polymer can also beprepared with mixtures of appropriate vinyl acylates.

In the crosslinking agent of the formula (I), A preferably represents abranched or unbranched aliphatic hydrocarbon radical of 2 to 5 carbonatoms, in particular 2 or 3 carbon atoms. This hydrocarbon radical isparticularly preferably an ethylene or propylene radical. If R₁ /R₂ ofthis formula (I) stand for 1-acyloxyvinyl or 2-acyloxyallyl, the acyloxygroup therein preferably contains 2 to 18 carbon atoms, in particular 2to 6 carbon atoms. Acyloxy preferably denotes an acetate or propionateradical. The radicals R₁ /R₂ preferably denote vinyl. A preferredcrosslinking agent unit in the polymer according to the invention iscorrespondingly derived from N,N'-divinylethylene urea. Thiscrosslinking agent brings about a particularly hydrolysis-resistant formof crosslinking. Another preferred representative isN,N',-divinylpropylene urea.

The preparation of these compounds is known and is described, forexample, in U.S. Pat. No. 2,541,152 and Ullmann, Encyklopadie dertechnischen Chemie [Encyclopedia of Industrial Chemistry], volume 23,611 (4th edition).

In the crosslinking agent of the formula (II), B preferably denotes adivalent hydrocarbon radical, in particular a branched or unbranchedalkylene radical of 2 to 6 carbon atoms, preferably 2 to 4 carbon atoms.In this instance the acyloxy group preferably has the same meaning as isdescribed above for the R radical in the formula (I). An example of apreferred crosslinking agent of this type is 3,3-dimethylpentadiene2,4-diacetate, which copolymerizes particularly readily with the vinylacylate. Compounds of this type can be prepared, for example, byreacting the appropriate diketone, triketone or tetraketone with vinylacylate or isopropenyl acylate in the presence of acid catalysts, thecorresponding enol acylates being formed. The acetone which is formed atthe same time has to be continuously removed out of the equilibriummixture by distillation.

The amount of units of crosslinking agent (II) generally accounts for 0to 100%, in particular 0 to 60%, of the total amount of crosslinkingagent units in the polymer.

The total amount of crosslinking agent units in the polymer according tothe invention is within the claimed ranges and depends on thecrosslinking density desired for the specific intended use. Forinstance, in gel chromatography a high dimensional stability isdesirable, which presupposes a high crosslinking density and hence ahigher content of crosslinking monomer units. By comparison, a lowercrosslinking agent density can be advantageous in other areas of use,for example as carrier material for enzyme reactions in a stirred vesselor for diagnostic agents. Crosslinking agent contents below 0.1% byweight, in most cases, no longer produce useful products. Crosslinkingagent contents above 60% by weight are basically possible, but, as arule, do not lead to further advantages.

Depending on the intended use, the amount of crosslinking agent units ispreferably 1 to 50% by weight, in particular 1 to 40% by weight, of thepolymer. For use as a carries material for biologically activesubstances, the lower limit is preferably 2.5% by weight, particularlypreferably 10% by weight. If only crosslinking agent units of theformula (II) are present, their lower limit is particularly preferably2.5% by weight.

For some uses, it can be of advantage for the polymer according to theinvention also to contain monomer units of a monomer which iscopolymerizable with vinyl acetate, their amount generally not exceeding10% by weight of the total polymer and preferably being between 0.1 and5% by weight. Examples of these monomer which may be used in the mixtureare: N-vinylpyrrolidone, vinylene carbonate, (meth)acrylic acid,(meth)acrylnitrile, (meth)acrylamide, alkyl (meth)acrylates each of 2 to12 carbon atoms, preferably 2 to 4 carbon atoms, in the alkyl radical,hydroxyalkyl esters of (meth)acrylic acid having 2 to 6 carbon atoms inthe alkyl group, N-vinyl-N-alkyl-acetamide, styrene, α-methylstyrene andthe like.

The crosslinked polymer according to the invention is preferably in theform of beads which are predominantly spherical in shape, whose averageparticle size in the dry, unswollen state is 20 to 800 μm, preferably 50to 300 μm, and which preferably have a narrow particle sizedistribution. The most suitable particle size depends in each case inthe main on the specific field of use. For instance, for a column methodcarried out in the absence of pressure an appropriately higher particlesize would be chosen, within the abovementioned limits, than for apressurized method. The beads of the polymer according to the inventionare constituted predominantly macroporously. The average pore diameteris generally within the range from 2 to 10,000 nm, preferably 5 to 200nm, in particular 20 to 200 nm.

The pore diameter (pore volume) can be determined by first of alldetermining the pore volume by the capillarly pressure method (mercuryporosimetry) (cf. in this context "Ullmanss Encyklopadie der technischenChemie" [Ullmann's Encyclopedia of Industrial Chemistry], volume 5(1980), pages 751-752). From the pore volume the average pore diametercan then be calculated by the equation given at the top of the left-handcolumn on page 752 of this reference. It is also possible to determinethe pore size by scanning electron microscopy.

The acylate groups of the vinyl acylate units in the polymer accordingto the invention have preferably been hydrolyzed into OH groups, thedegree of hydrolysis being generally more than 50%, preferably more than70% and in particular 90 to 100%. If the crosslinked polymer obtainedfrom hydrolysis, namely polyvinyl alcohol, is to be used as a carriermaterial for biologically active substances, preferably at least aportion of the OH groups are occupied by so-called spacer groups, whichare defined below. By contrast, for some purposes of gel chromatographyit can be of advantage for at least some of the OH groups to be occupiedby hydrophobing groups which no longer contain reactive radicals.

The polymers according to the invention are distinguished in particularby high resistance to hydrolysis combined with high crosslinkingdensity. This high resistance to hydrolysis is of great importance notonly in gel chromatography but also in the use as a carrier material forbiologically active substances, such as enzymes. Carrier-supportedenzymes are frequently employed for years in a strongly alkaline orstrongly acid medium. This is especially true of "nonspecifichydrolases", which cleave ester or carboxamide bonds. As for the rest,the stable crosslinking is also advantageous in the hydrolysis of theacylate groups into OH groups in the polymers according to theinvention.

The polymers according to the invention are suitable, inter alia, foruse as the stationary phase in gel chromatography and as a carriermaterial for biologically active substances.

The crosslinked polymers according to the invention are prepared inconventional manner, preferably under bead polymerization conditions inthe presence of a dispersion medium and of a dispersion stabilizer andin the absence or presence of further additives and in the absence orpresence of a free-radical initiator and preferably of an inert diluentand with the exclusion of oxygen.

Suitable dispersion media for carrying out the bead polymerization arein the main compounds which are liquid under normal conditions and havea boiling point of above 60° C., preferably within the range from 85° to300° C., and in which, under the polymerization conditions, themonomers, the polymer and preferably also the initiator are insoluble orat any rate only sparingly soluble, in order to suppress any emulsionpolymerization. The ratio of the monomer phase to the dispersion mediumphase can vary within wide limits, for example between 0.5:1 and 1:50,preferably 1:1 and 1:15 (by weight). According to the invention thepreferred dispersion medium is water. The water advantageously containsa buffer which operates within the alkaline range and makes the hydrogenion concentration resistant to the acid formed by hydrolysis of vinylacylate. This buffer preferably consists of Na₂ HPO₄ /NaH₂ PO₄ orNaHCO₃.

The dispersion stabilizer, which is to prevent the beads fromagglomerating in the course of the polymerization, can be one of thecompounds known for this purpose. It is preferably a hydrophilicpolymer, such as polyvinylpyrrolidone, polyvinyl alcohol,polyacrylamide, polyethylene glycol, methylcellulose or ethyleneoxide/propylene oxide copolymer. Polyvinylpyrrolidone is particularlypreferred for this purpose. These dispersion stabilizers are effectivein amounts as low as 0.001% by weight of the total amount of monomer.Usually the amounts used range from 0.005 to 50% by weight, preferably0.01-20% by weight (based on the total amount of monomer).

The addition of an electrolyte to the aqueous phase (if the dispersionmedium is water), for example of a salt such as sodium chloride, isgenerally advantageous, since this has the effect of almost completelydisplacing the monomer out of the outer phase and hence of almostcompletely stopping emulsions from forming and, additionally, ofincreasing the yield of beads. The added electrolyte, moreover, canalso, to some extent, have the action of a protective colloid. Thiselectrolyte is usually used in amounts of up to 50% by weight,preferably up to 30% by weight, based on the dispersion medium.

According to the invention, suitable free-radical initiators should bereadily soluble in the monomer phase and be as sparingly soluble aspossible in the liquid dispersion medium. Examples thereof are organicperoxides, such as di-tert.-butyl peroxide, dibenzoly peroxide, cumenehydroperoxide or cyclohexanone peroxide, and aliphatic azo compounds,such as α,α'-azodiisobutyronitrile, azobiscyanovaleric acid,1,1'-azocyclohexane-1,1'-dicarbodinitrile or azodicarboxamide.Appropriate redox systems can also be used. The amount of initiator isusually 0.01-5% by weight, preferably 0.1 to 2% by weight (based on thetotal amount of monomer). It is also possible to initiate thepolymerization by radiation in the absence or presence of an initiator.

To obtain as high a porosity as possible in the bead polymers, certaininert, liquid components (diluents) are added to the polymerizationsystem or preferably the monomers. For the purposes of the presentinvention, these diluents are substances in which the monomers arereadily soluble or with which the monomers are miscible, but which, onthe other hand, are virtually insoluble in the dispersion medium andhence are immiscible therewith. Diluents of this type and their mode ofaction are described, for example, in German Pat. No. 1,517,935 andMakromol. Chemie 176, pages 657 et seq. (1975).

The most suitable diluent or diluent mixture is readily determined by afew simple routine experiments. The pore size can be affected by thenature, composition and amount of the inert component, but it alsodepends on the amount of crosslinking component.

The diluents can be used either alone or mixed and be solvents orcoagulants for polyvinyl acetate. Examples are: alkanols, such asbutanol, cyclohexanol, isooctanol or glycol, esters, such as butylacetate, butylglycol acetate or glycerol triacetate, amides, such asdimethylformamide, dimethylacetamide or pyrrolidone, ketones, such asacetone or cyclohexanone, ethers, dialkyl ethers of at least 6 carbonatoms, such as di-n-butyl ether, di-n-amyl ether or diphenyl ether, andhydrocarbons, such as hexane, benzene, isooctane or paraffin oil. Thepreferred diluents, for water as the dispersion medium, are dialkylether of at least 6 carbon atoms, such as di-n-butyl ether or di-n-amylether. Other preferred diluents are polyglycols which are formed byaddition of a mixture of ethylene oxide and propylene oxide or ofpropylene oxide alone onto an alcohol, for example butanol as thestarting molecule, and which may have a random distribution of ethyleneoxide and propylene oxide or be block polymers of ethylene oxide andpropylene oxide where poly(oxyethylene) units are added to both ends ofthe poly(oxypropylene) chain.

The amount of added diluent is largely variable. It depends, inter alia,on the monomer composition, in particular the crosslinking agentcontent, on the desired porosity (pore size), and on the exact purposeintended for the polymer. Thus, in the case of a high degree ofcrosslinking it is advisable for the amount of diluent to be high aswell in order to obtain a certain porosity (pore size). Similarly, for agiven degree of crosslinking the porosity (pore size) will be higher thehigher the amount of diluent. Naturally, there are limits to the amountof diluent, since otherwise the mechanical strength becomesinsufficiently low. In most cases, satisfactory results are obtained ifthe volume of diluent is 0.02 to 5 times, preferably 0.04 to 3 times,the volume of monomer used.

The vinyl acylate, the crosslinking agent and the further comonomer(s)are used in such amounts as to produce a polymer containing theabovementioned levels of monomer units.

The process according to the invention is advantageously carried out attemperatures of usually 20°-150° C., preferably 20°-100° C., and under apressure of 1-10 bar, preferably 1-5 bar, in a reaction vessel equippedwith a stirrer. The particle size of the bead polymer is set inconventional manner by the stirrer speed and the phase ratio. Thereaction vessel is preferably vacuumtight and can be equipped withreflux condenser, addition funnel, gas inlet tube andtemperature-measuring means. The vessel is generally heated and cooledby means of a liquid bath, for example an oil bath or water bath. It isadvantageous to carry out the process according to the invention in theabsence of atmospheric oxygen. For this reason the reaction vessel isflushed before the start with an inert gas, preferably nitrogen.

After the polymerization reaction has ended, the unreacted monomers areremoved out of the reaction vessel, for example by vaporization underreduced pressure, preferably under a pressure of 0.1-15 mm Hg. After theresidual monomers have been removed, the dispersion medium is separatedfrom the solid polymer, for example by decanting, filtering or suckingoff the supernatant liquor. The diluent, if used, can be removedbeforehand by steam distillation. Afterwards the polymer is washed, ifnecessary, with low-boiling organic solvents, for example a hydrocarbon,a lower alcohol or acetone, and is finally dried at a temperature ofusually 20°-100° C., preferably 20°-80° C., ideally under reducedpressure.

The polyvinyl acetate gel thus obtained is not hydrophilic; the estergroup has to be hydrolyzed before use in water. The hydrolysis can becarried out in conventional manner under alkaline conditions by swellingthe product in an alcohol, such as, for example, methanol, and addingaqueous alkali, such as, for example, sodium hydroxide solution, or bytransesterifying the alcohol-swollen product with catalytic amounts ofacid or base while the ester formed is continuously removed by, forexample, distillation (cf. German Pat. No. 1,517,935). The hydrolysiscan be discontinued at any stage, so that the degree of hydrophilicityof the gel can be set in accordance with the intended use.

If the crosslinked polyvinyl alcohol gel in bead form is used as acarrier for biologically active substances which are to be fixed ontothe carrier by covalent bonding, it is in many cases advantageous tomodify the gel beforehand with so-called spacers. For the purposes ofthe present invention, spacers are compounds which react both with thesupport polymer and the biologically active substance and form a bridge,as it were, between the two. The reaction of the bead polymer with thespacer can take place either directly or preferably after priorhydrolysis of the acylate groups. The degree of conversion depends,inter alia, on the bulkiness of the spacer and the accessibility of theacylate group or of the secondary hydroxyl groups formed therefrom.According to the invention, suitable spacers are the homo- andhetero-bifunctional compounds which are known for this purpose and whosesecond functional group takes on the coupling to the biologically activesubstance to be fixed (cf. German Pat. Nos. 2,421,789 and 2,552,510,Ullmanns Encyclopadie der technischen Chemie [Ullmann's Encyclopedia ofIndustrial Chemistry], 4th Edition, Volume 10, page 540 and"Characterization of Immobilized Biocatalysts", Verlag Chemie, Weinheim,1979, page 53).

Examples of spacers used according to the invention are those whichintroduce the following groups: ##STR5##

According to the invention, preferred spacers bring abouthydrolysis-resistant chemical bonds and include epichlorohydrin or itshomologs (α,κ-epoxy-ω-halogenoalkanes). The reaction of the polyvinylalcohols (polyvinyl acylates) takes place in the absence or presence ofa solvent, preferably in the presence of a catalyst. The reactiongenerally takes between 30 minutes and 24 hours, depending on thetemperature, which can be between room temperature and the refluxtemperature of epichlorohydrin (113°-115° C.). Examples of suitablecatalysts are NaOH (in powder form), aqueous alkalis, dimethylformamide,triethylamine and other acid acceptors.

For the purposes of the present invention, biologically activesubstances are known natural or artificial substances of in vivo or invitro activity, such as enzymes, activators, inhibitors, antigens,antibodies, vitamins, hormones, effectors, antibiotics, proteins and thelike. Proteins also include proteins having certain nonproteinsubstituents, such as metal ions, polysaccharides, porphyrin groups,adenine dinucleotide, ribonucleic acid, phospholipids and so on.Biologically active substances even include polypeptide fragments, forexample the active parts of enzyme molecules.

According to the invention, enzymes are preferred among theabovementioned biologically active substances. Examples of enzymes areurease, penicillin acylase, D-aminoacid oxidase, adenyl deaminase,alcohol dehydrogenase, asparaginase, carboxypeptidase, chymotrypsin,diphosphoesterase, α-glucosidase, glucose isomerase, glucose oxidase,glucose-6-phosphate dehydrogenase, hexokinase, invertase, κ-lactamase,lactase, lactate dehydrogenase, various lectins, NAD kinase,neuraminidase, papain, peroxidase, alkaline and acid phosphatases,5'-phosphodiesterase, pyruvate kinase, ribonuclease and trypsin.

Examples of other biologically active substances are hormones, such asinsulin and various pituitary hormones, proteins of the gamma-globulinfraction, for example antibodies of classes G, M, A, D and E, otherblood factors, for example antihemophilic factor, the blood-clottingfactors, specific antibodies, for example hepatitis, poliomyelitis,measles, mumps, influenza or rabbit antibodies, antigens, such ashepatitis, polyomyelitis, measles, mumps, influenza or rabbit antigensfor purifying or stimulating suitable antibody reactions in which theantigen (once insolubilized) remains in the insoluble form andconsequently cannot penetrate and damage the body, as well as generalbody proteins, such as hemoglobin or albumin.

The anchoring reaction with the biologically active substance is carriedout in conventional manner, as described for example in GermanOffenlegungsschrift 2,407,340 or German Pat. Nos. 2,215,687, 2,421,789and 2,552,510. The reaction usually takes place at room temperature,i.e. at +40° C. or below, the latter in particular when the biologicallyactive substance to be anchored is unstable by nature; in this case thetemperatures are then below +10° C., preferably at 0° to +5° C.

The anchoring reaction preferably takes place at around a neutral pH,for example pH 5-9, since in this pH range most of the biologicallyactive substances are at their most stable. Nor, as a rule, is itnecessary to maintain more strongly acid or alkaline conditions, sincethe macroporous bead polymers undergo rapid reaction with most of thesubstances in question even in the neutral range. The resulting bondaffords sufficient stability for long storage periods and high stabilityin operations.

The invention is illustrated in more detail by the following Examples.

EXAMPLE 1

An organic phase comprising a solution of 97.5 g of vinyl acetate, 2.5 gof divinylethyleneurea and 0.1 g of azoisobutyronitrile was suspendedwith stirring in an aqueous phase comprising 4.2 g of Na₂ HPO₄, 0.25 gof NaH₂ PO₄, 7.0 g of polyvinylpyrrolidone and 700 ml of H₂ O undernitrogen in a flask equipped with a stirrer, a thermometer and a refluxcondenser. The polymerization was started by heating to 75° C. by meansof a hot bath. After two hours the temperature was raised to 85° C. Afurther two hours later the polymerization was complete. The suspensionobtained was cooled down by pouring into ice, the finely dispersedemulsion was repeatedly decanted off, and the polymer was filtered offand dried. The amount of dry product obtained was 80 g.

To hydrolyze the product, 50 g thereof were swollen in methanol, and asolution of 50 g of NaOH in H₂ O was added at 25° C. without heating orcounter-cooling. After twelve hours the product was filtered off, waswashed with plenty of water until neutral, and was dried.

The products were used in gel chromatography. The unhydrolyzed gel wasfound to have an exclusion molecular weight of 1,200 for polystyrene intetrahydrofuran. The hydrolyzed product had an exclusion molecularweight of 1,100 for polyethylene glycol in water.

EXAMPLE 2

The polymerization of Example 1 was carried out in the presence of 140 gof NaCl in the aqueous phase. When the reaction was ended by pouring thesuspension into ice-water, all the bead polymer immediately settled out,so that almost no emulsified content was obtained. The isolatable yieldof bead polymer was above 90% (based on the polymerizable phase).

The exclusion molecular weight of the gel for polystyrene intetrahydrofuran, rose to 1,500; after the hydrolysis the exclusionmolecular weight was found to be 1,300 for polyethylene glycol in water.

EXAMPLE 3

The polymerization of Example 1 was repeated, except that thecrosslinking component comprised 2.0 g of divinylethyleneurea and 0.5 gof 3,3-dimethylpentadiene 2,4-diacetate.

The gel chromatography data corresponded to those of Example 1.

The 3,3-dimethylpentadiene 2,4-diacetate was prepared as described belowby acylating 3-methylbutan-2-one with acetic anhydride in the presenceof a Lewis acid and subsequently reacting the resulting diketone withisopropenyl acetate:

330 g (3.84 mol) of freshly distilled 3-methylenebutanone were mixedwith 500 g (5 mol) of technical acetic anhydride (95%) in a flask, and160 g (1.15 mol) of ZnCl₂ were added with stirring under a stream ofnitrogen. The contents of the flask were heated at 120° C. for 3 hours,were cooled down, and were distilled in a water jet vacuum.

This produced 363 g of a crude product having a boiling point of 62° C.(12 mm Hg) and a GC purity of 86%.

Renewed distillation of this substance in a water jet vacuum produced289 g of a uniform product having a boiling point of 60° C. (12 mm Hg)which, on ¹ H-NMR analysis, proved to be 3,3-dimethylpentane-2,4-dione(singuletts at 1.3 ppm and 2.05 ppm).

110 g of 3,3-dimethylpentane-2,4-dione were mixed with 500 g of dryisopropenyl acetate under nitrogen, and 5 g of p-toluene sulfonic acidwere added. The mixture was heated to the reflux temperature under ashort packed column, and for several days distillate fractions weretaken off at between 54° C. and 90° C. for brief periods at 12-hourintervals over several days. The composition of the reaction mixture wasmonitored by gas chromatography. After the reaction period of 6 days wasover, the acid catalyst was neutralized by adding carbonate, and thereaction mixture was quickly distilled off in a water jet vacuum.Renewed distillation under atmospheric pressure served to removeunreacted isopropenyl acetate. Renewed distillation under a water jetvacuum produced 39 g (87.5° C./12 mm Hg) of a 100% pure substance by gaschromatography. This product was 3,3-dimethylpent-1-en-4-on-2-yl acetate(¹ H-NMR: Singulett at 1.3 ppm and 2.1 ppm, Duplett at 4.9 ppm).

On further distillation the remainder of the crude product producesunder an oil pump vacuum of 0.01 mm Hg a product mixture of a monoenolacetate and a second, higher-boiling compound. This produced, between54° C. and 65° C. under 0.01 mm Hg, an additional 15 g of a dienolacetate in a purity of 93% (GC).

EXAMPLE 4

To carry out a heterogeneously crosslinking bead polymerization, asolution of 80 g of vinyl acetate, 20 g divinylethyleneurea, 1 g ofazoisobutyronitrile and 200 g of n-heptanol was dispersed andpolymerized in a solution of 0.175 g of NaH₂ PO₄, 3 g of Na₂ HPO₄ and 5g of polyvinylpyrrolidone in 500 ml of water. The temperature variationcorresponded to that of Example 1. After four hours the diluent wasremoved by steam distillation, and the product was isolated. The yieldwas 77.7 g of completely spherical clear bead polymer. The averageparticle diameter was about 30 μm (stirrer speed 460 rpm).

The product had a bulk volume of 1.55 ml/g. In tetrahydrofuran its gelbed volume was 5.77 ml/g and the exclusion molecular weight forpolystyrene was 80,000. The hydrolyzed product had a bulk volume of 1.54ml/g, which rose on swelling in water to 5 ml/g, and had an exclusionmolecular weight for polyethylene glycol of 20,000.

20 g of the hydrolyzed bead copolymer were allowed to swell at roomtemperature in 200 ml of epichlorohydrin for 24 hours. The temperaturewas then raised with slow stirring to 113°-115° and maintained for 4hours. After cooling down the mixture was filtered with suction, and thecopolymer was repeatedly triturated for 1-hour periods at a time inacetone. The acetone-moist copolymer was dried at 50° C. in a vacuumcabinet to a constant weight. The epoxy equivalent weight was 244 (asmeasured by Axen method: Acta Chem. Scand. Volume 29 (1975) No. 4).

EXAMPLE 5

The polymerization of Example 4 was repeated, except that the diluentwas replaced by a mixture of 80 g of 2-ethylhexanol and 20 g of apolyglycol of ethylene oxide and propylene oxide (weight ratio 1:1;random distribution) having a molecular weight of about 1,200 andobtained by adding ethylene oxide and propylene oxide onto butanol asthe starter. ("Polyglykol B 11/50" from HOECHST AG).

On isolation this produced 76 g of a chalk-white spherical bead polymerwhose particle size was 50 to 200 μm at a stirrer speed of 460 rpm.

EXAMPLE 6

The polymerization of Example 4 was repeated, except that the diluentwas replaced by a mixture of 70 g of 2-ethylhexanol and 30 g of apolyglycol having a molecular weight of about 700 and obtained by addingpropylene oxide onto butanol as the starter ("Polyglykol B 01/20" fromHOECHST AG).

On isolation this produced 82 g of a chalk-white spherical bead polymerwhose particle size was 50 to 200 μm at a stirrer speed of 460 rpm.

EXAMPLE 7

The polymerization was repeated, except that the diluent was replaced bya mixture of 80 g of 2-ethylhexanol and 20 g of a polyglycol of ethyleneoxide and propylene oxide (weight ratio: 4:1; random distribution;molecular weight: about 5,000) obtained by adding ethylene oxide andpropylene oxide onto butanol as the starter ("Polyglykol P 41/300" fromHOECHST AG).

On isolation this produced 68 g of a chalk-white spherical bead polymerwhose particle size was within the range from 50 to 200 μm at a stirrerspeed of 460 rpm.

EXAMPLE 8

The polymerization of Example 4 was repeated, except that the diluentwas replaced by a mixture of 80 g of 2-ethylhexanol and 20 g of apolyether glycol of polyoxypropylene and polyoxyethylene having 10% byweight of polyoxyethylene in the total molecule, the polyoxyethyleneunits having been added to both ends of the polyoxypropylene chain(molecular weight: about 1,750) ("Pluronic polyol 61" from BASF,Wyandotte Corp.).

On isolation this produced 72 g of a chalk-white spherical bead polymerwhose particle size was in the range from 50 to 200 μm at a stirrerspeed of 460 rpm.

EXAMPLE 9

The polymerization of Example 4 was repeated, except that the diluentwas replaced by a mixture of 100 g of 2-ethylhexanol and 100 g ofdi-n-butyl ether. On isolation this produced 83 g of a chalk-whitecompletely spherical bead polymer whose particle size was 70 μm at astirrer speed as in Example 4.

The bulk volume of the product was 2.81 ml/g, and in tetrahydrofuranthis had a gel bed volume of 7.48 ml/g and an exclusion molecular weightfor polystyrene of 2×10⁶. The hydrolyzed product had a bulk volume of1.6 ml/g, the gel bed volume in water was 12.8 ml/g, and the exclusionmolecular weight for polyethylene glycol was 2×10⁶.

EXAMPLE 10

The polymerization of Example 4 was repeated, except that the dispersedphase comprised 70 g of vinyl acetate, 30 g of divinylethyleneurea, 1 gof azobisisobutyronitrile and 158 g of di-n-butyl ether. This produced77 g of a white bead polymer having an average diameter of 200 μm.

The bulk volume was 2.9 ml/g, and the gel bed volume in tetrahydrofuranwas 7.45 ml/g. The exclusion molecular weight of the product could notbe determined by gel chromatography: polystyrene having a molecularweight of 25,000,000 was eluted with almost the internal volume.Scanning electron micrographs showed pores of more than 100,000 Å indiameter.

The hydrolyzed product had a bulk volume of 6.5 ml/g, which showed thatthe skeleton structure had been completely preserved. The gel bed volumein water was 14.5 ml/g; in the analysis by gel chromatography,polyethylene glycol having a molecular weight of 3.8×10⁶ had access toalmost the entire internal volume.

10 g of the hydrolyzed copolymer were allowed to swell in 100 g ofepichlorohydrin for 24 hours, and the temperature was then graduallyraised with stirring to 110° and maintained for 12 hours. After coolingdown to room temperature the mixture was filtered off with suction, andthe copolymer was repeatedly stirred out slowly in acetone for 2 hoursat a time. The drying took place at 50° C. in a vacuum cabinet. Theepoxy equivalent weight was 105 μmol per g of carrier substance.

Reaction of the bead-shaped polymer carrier according to the inventionwith biologically active substances EXAMPLE 11

800 μl of a trypsin solution (6.25 mg/ml, 345 U/ml) were added to 100 mgof a carrier prepared as in Example 4. 1M potassium phosphate buffer wasadded to bring the enzyme solution to pH 7.8, and 1.6×10⁻² M benzamidinesolution was added to stabilize the active center of the enzyme. Theduration of fixing of the enzyme to the carrier was 72 hours at 25° C.The trypsin not bonded covalently to the carrier was then filtered offwith suction via a glass fritte, and the residue was washed repeatedlywith 1M sodium chloride solution and then with buffer solution. Theyield of moist material on the filter was 324 mg. The measurement wascarried out at 37° and pH 7.8 with an autotitrator usingN'-benzoyl-L-arginine ethyl ester hydrochloride (BAEE) and produced avalue of 227.5 U/g in the moist state or 356 U/g based on the dryweight. From the starting and wash water activities the fixation yieldcould be calculated as 20%.

EXAMPLE 12

1500 μl of a urease solution (30 mg/ml, 51 U/ml) brought to pH 8.0 with1M potassium phosphate buffer were added to 200 mg of an epoxidizedcarrier prepared as in Example 10. After a period of fixation of 16hours at room temperature the carrier was repeatedly washed with 1Msodium chloride solution and then with buffer solution. The yield ofmoist carrier on the filter was 754 mg. The measurement with anautotitrator at 30° and pH 8.0 using urea as substrate revealed anactivity of 100 U/g (moist) or 377 U/g based on the dry weight of thecarrier. From the starting and wash water activities it was possible tocalculate a fixation yield of 98%.

We claim:
 1. An absorbent in chromatography or a carrier material forbiologically active substances comprising a crosslinked polymer whichconsists essentially of (a) vinyl acylate units and (b) units of atleast one crosslinking agent of the formula ##STR6## in the presence orabsence of (c) units derived from an additional monomer which iscopolymerizable with (a) wherein R₁ and R₂ in the formula (I) areidentical or different and each denotes vinyl-, 1-acyloxyvinyl, allyl-,or 2-acyloxyally, A represents a divalent hydrocarbon radical of 2 to 8carbon atoms, the units of the crosslinking agent accounting for 0.1 to60% by weight of the polymer and wherein the acylate groups of the vinylacylate units are at least partially hydrolyzed.
 2. The absorbent orcarrier material as claimed in claim 1, wherein said polymer containsvinyl acetate units.
 3. The absorbent or carrier material as claimed inclaim 1, wherein the acyloxy group in the radicals R₁ and R₂ of theformula (I) has 2 to 6 carbon atoms.
 4. The absorbent or carriermaterial as claimed in claim 1, wherein the units of crosslinking agentaccount for 1 to 50% by weight of said polymer.
 5. The absorbent orcarrier material as claimed in claim 1, wherein at least 10% by weightof the acyloxy groups of the vinyl acylate units are replaced byhydroxyl groups.
 6. A process for preparing a crosslinked polymerconsisting essentially of vinyl acylate units and units of at least onecrosslinking agent of the formula ##STR7## wherein R₁ and R₂ in theformula (I) are identical or different and each denotes vinyl,1-acyloxyvinyl, allyl or 2-acyloxyallyl, A represents a divalenthydrocarbon radical of 2 to 8 carbon atoms, the amount of thesecrosslinking agent units accounting for 0.1 to 60% by weight of thepolymer comprising the steps of: bead polymerizing (i) vinyl acylatewith (ii) a crosslinking agent of formula (I) in the presence or absenceof (iii) an additional monomer which is present in an amount notexceeding 10% by weight of the total polymer, and which iscopolymerizable with the vinyl acylate, said bead polymerizing stepoccurring at a temperature of 20° C. to 150° C. and under a pressure of1 to 10 bar in the presence of (iv) a dispersion medium; (v) at leastone inert diluent present in a volume of 0.02 to 5 times the volume ofmonomers used, (vi) a dispersion stabilizer and in the absence orpresence of (vii) a further additive and (viii) a free-radical initiatorwith the exclusion of oxygen, andhydrolyzing at least partially theacylate groups of the resulting polymer.
 7. The process as claimed inclaim 6, wherein the vinyl acylate is vinyl acetate.
 8. The process asclaimed in claim 6, wherein the dispersion medium is an alkaline aqueousbuffer solution.
 9. The process as claimed in claim 6, wherein thedispersion medium contains 0-50% by weight of an electrolyte.
 10. Theprocess as claimed in claim 6, wherein the dispersion medium contains anonionic surfactant as the dispersion stabilizer.
 11. The process asclaimed in claim 6, wherein the polymerization step is carried out inthe presence of a dialkyl ether of at least 6 carbon atoms as the inertdiluent.
 12. The process as claimed in claim 6, wherein the acyloxygroup in the radicals R₁ and R₂ of the formula (I) has 2 to 6 carbonatoms.
 13. The process as claimed in claim 6, wherein the units ofcrosslinking agent account for 1 to 50% by weight of said polymer. 14.The process as claimed in claim 6, wherein at least 10% by weight of theacyloxy groups of the vinyl acylate units are replaced by hydoxylgroups.
 15. The process as claimed in claim 6, wherein the hydrolysis iscarried out by swelling the resulting polymer in an alcohol, and addingan aqueous alkali or by swelling said resulting polymer in an alcoholand transesterifying said polymer with catalytic amounts of acid orbase.
 16. An adsorbent for chromatography obtained from the crosslinkedpolymer as claimed in claim
 1. 17. The polymer as claimed in claim 1 inthe form of macroporous beads with an average particle size of from 20to 800 μm and an average pore diameter of from 2 to 10,000 nm.